The invention relates generally to tissue micro array processing and imaging.
Tissue micro arrays (TMA) are used for many analytic and diagnostic purposes, one of which is to diagnose diseased tissue at the molecular level. Whatever the purpose, tissue micro arrays, on which tissue samples are fixed, are typically stained with a morphological stain or biomarker and then analyzed manually with a microscope, or an image may be taken of the TMA to retain the image for subsequent analysis or comparison. After the first stain is applied and imaged, one or more serial or successive stains or biomarkers may be applied and the TMA is analyzed again. The two or more serial images may then be compared. It has been an ongoing goal to automate this system while maintaining the quality and consistency of the analysis and resulting conclusions or data. Such efforts have proved to be less than optimal because of the inability to automate the analysis of the TMA after each serial stain is applied. Although systems exist that attempt to combine the serial images, these combined images result in inconsistent and inconclusive results because the serial images are merely co-registered based on the mechanical placement of the TMA on the imaging microscope. These combined images that are co-registered based solely on the mechanical location of the TMA fail to incorporate differences between the serial images including, but not limited to, mechanical misplacement, tissue distortion, autofluorescence, differing levels of focus when each image is taken, and anomalies associated with the curvature of the cells in the tissue sample.
Pathologists have used hematoxylin-eosin (H&E) staining for over a hundred years. Hematoxylin stains cell nuclei blue, while eosin, as a counter-stain, stains cytoplasm and connective tissue pink. In view of the long history of H&E use, well-established methods of use, and the large amount of data and publications relating to H&E use, there is a strong belief among many pathologists that H&E will continue to be the common practice over the next fifty years. However, more recently developed technologies, that use molecular biomarkers to obtain functional information and sub-cellular localization, have also proved to be valuable for diagnostics, prognosis and survival rates for various diseases.
H&E staining techniques are often favored because they are generally low cost, fast and efficient; the images are readily acquired and there exists a large body of knowledge and training about these techniques. On the other hand, molecular biomarkers can provide protein-associated pathways that are not visible with H&E techniques. More recently, immunohistochemistry (IHC) based image analysis algorithms were developed to quantify the localization of proteins in the tissue. The value of these more recent techniques generally exceeds the value of the H&E techniques in terms of survival rates, prognosis, population segmentation and drug response prediction. Nevertheless, due to the common use of H&E and the availability of data with known outcome and diagnosis associated with H&E, most image analysis and automated quantification techniques still rely primarily if not entirely on H&E data.
In instances in which H&E techniques and molecular biomarkers are used, the current pathological tissue imaging modalities involve either molecular labeling or traditional H&E labeling but not simultaneous labeling. Simultaneous labeling of both techniques is not used because of the auto-fluorescence characteristics of the H&E dyes. It is not possible to simultaneously image H&E dyes with molecular biomarker using current imaging techniques because of the auto-fluorescence characteristics of the H&E dyes. Chemical interactions of H&E dyes with the antibodies to which the molecular biomarkers are attached significantly limit the simultaneous imaging of H&E with biomarkers. As noted, although serial sections of H&E and fluorescent images have previously been serially compared, registration between such serial sections is poor because of tissue distortion and the optical and chemical effects of the stains.